The invention relates to a method of virus manipulation; means therefor and products thereof which have particular, but not exclusive, application in gene therapy.
Gene therapy of many diseases is now theoretically possible, as a result of recent advances in human genetics. The primary goal is the conversion of cell phenotype from a diseased to a normal state, through the delivery of trans-dominant acting genetic material. The conversion of this technology from cell culture systems to in vivo experimental models (and subsequently to the clinic) requires the development of new methods for efficient gene delivery in a controllable manner. It is becoming evident that whilst human genetics is moving at a rapid rate in the identification of disease-specific mutations, there is a relative lack of gene delivery system development. At present, there is a choice of either liposome, DNA aggregate or virus-based systems.
Liposome delivery is still very inefficient in DNA transfer (1), DNA aggregates formed between virus particles and charged materials such as polylysine do enhance DNA uptake (2) but standardisation of preparations is very difficult. Retrovirus and adenovirus vectors both have constraints in the size of heterologous DNA incorporated in the vector (3,4) and are unreliable in achieving long-term heterologous gene expression. Retroviruses integrate into the host genome but are difficult to produce as high titre stocks and have an inherently high rate of mutation through errors introduced during reverse transcription. Despite their broad cell tropism, adenoviruses induce a cell-mediated immune response and the nucleic acid is not stable long-term in infected cells (5).
Herpesviruses represent promising candidates for development as vectors, in part due to their ability to maintain their genome in cells in an episomal form which is blocked from replication. Their capacity for packaging heterologous DNA sequences is potentially  greater than 50 Kbp (6) and most are easy to manipulate in vitro. Herpes simplex derived vectors are likely to have some of the same problems as adenoviruses, in that the majority of the population already have a well-developed immune response to the virus. Other non-human herpesviruses which are capable of infecting human cells, however, should not suffer this disadvantage.
Herpesvirus saimiri (HVS) is a lymphotropic rhadinovirus (xcex32 herpesvirus) of squirrel monkeys (Saimiri sciureus). The virus may be routinely isolated from peripheral lymphocytes of healthy monkeys and causes no apparent disease in the species. The virus genome may be detected in an episomal form in T cells and genome transcription appears limited to three genes in the non-lytic (xe2x80x9clatentxe2x80x9d) state. The complete virus genome has been sequenced and shares many features in common with the human Epstein-Barr virus (EBV). The genetic organisation consists of a single unique coding region of DNA, 112.930 bp in length, flanked by a variable number of non-coding repeat sequences. There are 76 open reading frames, 60 of which have similarities with genes found in other herpesviruses (7). The remaining genes share sequence homology (at the level of protein) with human genes of known function, including complement control proteins, cell surface antigen CD59, cyclin D and G protein-coupled receptors (8, 9).
The virus has been divided into three distinct strains termed A, B and C based on their inability (A and B) or ability (C) to be oncogenic in certain other monkey species. C strains have the ability to transform human T cells to limited independent growth in vitro (10). This ability to transform cells is due to a gene termed STP (11) which has marked variability in protein sequence between strains such that only STP from C strains is able to transform cells (12). STP is not important for the normal lytic cycle of the virus or episomal maintenance and natural deletion mutants for this region of the virus genome exist (13); these strains are not oncogenic. Virus strains which lack this gene have been constructed which express selectable drug resistant markers (14). These viruses have been used to demonstrate that they are capable of infecting a wide range of human cell types, transferring heterologous genes with high efficiency and maintaining long-term expression in the absence of selective pressure. There is no evidence that this virus is able to produce any disease in man, although it is capable of infecting human cells. Thus it is likely that this virus represents a good starting point for the development of a non-replicating, safe vector for human cells. There is however a lack of basic understanding of how HVS replicates, particularly regarding transcriptional control and DNA replication.
Of all herpesviruses sequenced so far, HVS has the most homology with EBV. However the coding region is significantly smaller. Distinct gene blocks appear to be closely related between these two viruses, and indeed the herpesviruses in general. HVS differs from other herpesviruses due to the presence of certain genes which have not been identified in any other herpesvirus to date. Every virus vector in human trials to date has been disabled either through the deletion of genes which are non-essential for growth in culture or the deletion of essential genes and their provision in trans from helper cell lines. Extrapolation from the well studied herpesviruses allows us to predict that deletion of certain HVS membrane proteins will prevent cell-cell spread. Furthermore, the inactivation of proteins which control essential transcriptional switches, such as E1A in adenoviruses (17) and IE 175 in herpes simplex (18) will inevitably make such viruses replication incompetent. Thus, a major aim of this application is focussed on the construction of mutant viruses which are unable to activate early and late gene expression. The target genes are the two transcriptional control proteins which are the products of ORF 50 and 57, and likely to be essential for growth in tissue culture.
Published data (14) indicates that Strain 11/S4-derived viral vectors are only capable of limited growth in certain cell lines. Thus the need to delete, block or manipulate transcriptional control protein genes should only be necessary in cell lines that support viral replication. However, it may be desirable, in order to produce a virus for the purpose of gene delivery which one can use confidently, to produce a virus which is either unable to produce or which produces non functional transcriptional control proteins.
It is also another major aim of this application to identify genes which are non-essential for growth and then delete at least a part of at least one of these genes in order to facilitate the insertion of heterologous genetic material into the viral genome.
There currently exists a plasmid designed for recombination with herpesvirus saimiri which plasmid is designed to insert heterologous genetic material into the viral genome at a predetermined location, the location being the junction between the single unique coding region of DNA and a non-coding repeat sequence of herpesvirus DNA. However the plasmid is relatively inflexible in terms of what can be cloned into the viral genome. For example, there are few suitable restrictions sites and therefore the plasmid is not suitable for use commercially. We have therefore aimed in this application to identify non-essential genes for growth with a view to deleting at least a part of at least one of said genes with a view to providing artificial cloning sites for the insertion of large amounts of any selected heterologous genetic material. It will be apparent that the said deletion of non-essential genes and the subsequent insertion of heterologous genetic material will most advantageously be undertaken when large amounts of heterologous genetic material are to be inserted into the viral genome.
We aim in another aspect of our application to provide herpesvirus saimiri which has been manipulated so as to delete at least a part of at least one transcriptional control gene and, ideally, also at least a part of at least one gene that encodes a non-essential growth protein. We favour this aspect because the greater the number of viral genome manipulations the greater the safety of the manipulated virus. In view of this fact we also favour manipulation of the herpesvirus saimiri genome to bring about deletion, partially or wholly, of the STP gene. We favour this latter manipulation even in the instance where Strains A or B are to be utilised because we consider such a manipulation to increase the likely safety of the resultant manipulated virus.
It will be apparent from the above that there is a need to provide a suitable gene delivery system to enable intra-cellular delivery of genetic material which delivery is undertaken safely and thus without any cytopathological consequences at least on the target cell.
It is therefore a first object of the invention to provide a gene delivery system which is safe and controllable.
Furthermore, in view of the amount of genetic material likely to be delivered it is also an object of the invention to provide a gene delivery system which is adapted to accommodate large amounts of genetic material such as DNA sequences of 4 Kbp and up to 20 Kbp and, ideally,  greater than 50 Kbp.
It is a further object of the invention to provide a gene delivery system which allows selective recombination of at least a given gene, or part thereof, into same so as to deliver at least said selected gene, or part thereof, to a target cell.
In its broadest aspect the invention concerns the provision of mutant viruses which are unable to activate early and late gene expression. In other words it concerns the provision of a virus which is unable to replicate in a target cell and more preferably in human cells and/or the provision of mutant viruses which are adapted to accommodate relatively large amounts of heterologous genetic material.
According to a first aspect of the invention there is therefore provided a herpesvirus saimiri which has at least one mutation in a gene involved in virus replication whereby the mutation is such to prevent the virus replicating in a target human cell.
In a preferred embodiment of the invention said gene is either one or both of the transcriptional control protein genes ORF 50 and/or ORF 57.
Preferably further still said mutation comprises partial or complete deletion of one or both of said genes.
In yet a further embodiment of the invention the said herpesvirus saimiri is a strain either lacking or having a mutation in the STP gene so that the virus is unable to transform a target cell and so is unable to produce an oncogenic phenotype.
Preferably said virus is further manipulated so that at least a part of at least one gene encoding a non-essential growth protein is deleted. Ideally said gene is ORF4, ORF14, ORF15, ORF16 or ORF51.
In yet a still further preferred embodiment of the invention said virus is provided with an insertion site into which selected heterologous material can be inserted. Preferably the virus is manipulated so that insertion occurs either within, adjacent, or remote from, a deletion site for the deletion of at least a part of a non-essential growth protein gene; or in or adjacent at least one non-coding repeat sequence and more preferably at the junction between the single unique coding region of DNA and a non-coding repeat sequence. More preferably still, said virus is manipulated so that only one of said non-coding repeat sequences is present at one or both ends of the single unique coding region.
In the instance where insertion occurs within or adjacent said deletion site AND said deletion concerns either partial or whole deletion of one or more of the following genes ORF4, ORF14, ORF15, ORF16 or ORF51.
According to a yet further aspect of the invention there is provided a herpesvirus saimiri which has at least one mutation in at least one gene encoding a non-essential growth protein.
In a preferred embodiment of the invention said gene is either one or more of ORF4, ORF14, ORF15, ORF16 or ORF51.
Preferably further still said mutation comprises partial or complete deletion of one or more of said genes.
In yet a further preferred embodiment of the invention the said herpesvirus saimiri is a strain either lacking or having a mutation in the STP gene such that the virus is unable to transform a target cell and so is unable to produce an oncogenic phenotype.
Preferably said virus is further manipulated so that at least a part of at least one gene involved in virus replication is deleted. Ideally said gene is ORF50 and/or ORF 57.
In yet a further preferred embodiment of the invention said virus is provided with an insertion site into which selected heterologous material can be inserted. Preferably the insertion site is within, adjacent, or remote from, the site of said deletion of one or more of said genes.
According to yet a further aspect of the invention there is provided a herpesvirus saimiri either having therein or adapted to have inserted therein at least one preselected heterologous genetic fragment adjacent a deletion site which deletion site represents a site for partial or whole deletion of at least one gene encoding a non-essential growth protein.
In a preferred embodiment of the invention said virus is also provided with a mutation in a gene involved in viral replication so as to prevent viral replication following insertion of said virus into a target cell.
More preferably said virus is a strain either lacking or having a mutation in the STP gene such that the virus is unable to transform a target cell and so is unable to produce an oncogenic phenotype.
According to a further aspect of the invention there is provided a herpesvirus saimiri either having therein or adapted to have inserted therein at least one preselected heterologous genetic fragment at the junction of the single coding region and a non-coding region and further wherein said has viruses been manipulated so that only a reduced number of non-coding repeat sequences is present at one or both ends of the single coding region and there is also provided a mutation in a gene involved in viral replication so as to prevent viral replication following insertion of said virus into a target cell.
Preferably said number of non-coding repeat sequences is 5 or less and ideally one.
According to a yet further aspect of invention there is provided a transfer vector which enables insertion of a heterologous genetic fragment into herpes saimiri virus DNA.
Preferably said insertion involves any one or more of the afore described methods of insertion. In a preferred embodiment of this aspect of the invention said vector includes a plurality of unique restriction sites and more preferably three unique restriction sites. In addition, said vector includes a beta-galactosidase gene which is preferably under the control of the HCMV IE 3 promoter. More preferably the said vector is derived from pRUNeo (16) and ideally is prupoly.
According to yet a further aspect of the invention there is provided a herpesvirus saimiri which has at least one mutation in a gene involved in virus replication whereby the mutation is such to prevent the virus replicating in a target cell and also at least one mutation in a gene encoding a non-essential growth protein.
In a preferred embodiment of the invention said herpesvirus saimiri also has a mutation in the STP gene.
Preferably said mutations comprise either partial or complete deletion of said genes.
Preferably further still said gene involved in virus replication comprises either one or both of the transcriptional control protein genes ORF50 and/or ORF57: and said gene encoding a non-essential growth protein is one or more of the following genes: ORF4, ORF 14, ORF15, ORF16 or ORF51.
It will be apparent from the above that the preferred virus of the invention comprises a number of advantageous combinations of genetic mutations which combinations serve to disable and enable the virus so as to make it safe and controllable. By the term disable we mean the prevention of viral replication in a target cell and by the term enable we mean the capacity to accommodate the insertion of a relatively large amount of heterologous genetic material. More desirably still, said advantageous combination also provides for a virus unable to transform a target cell and so unable to produce an oncogenic phenotype.
According to a yet further aspect of the invention there is provided a target cell including at least a part of the herpesvirus saimiri gene therapy vector.
According to a yet further aspect of the invention there is provided a cell transformed with a herpesvirus saimiri vector as afore described.
According to a yet further aspect of the invention there is provided a method of delivering selected heterologous genetic material to a target cell comprising exposing at least said target cell to a herpesvirus saimiri which includes at least said preselected heterologous material under conditions which favour infection of said cell with said virus.